Stable aqueous alfa interferon solution formulations

ABSTRACT

Stable aqueous solution formulations containing alfa-interferon type interferon, e.g., interferon alfa-2a and interferon alfa-2b, a buffer to maintain the pH in the range of 4.5-7.1, polysorbate 80 as a stabilizer, edetate disodium as a chelating agent, sodium chloride as a tonicity agent, and m-cresol as an antimicrobial preservative and which maintain high chemical, physical and biological stability of the alfa-type interferon for an extended storage period of at least 24 months are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 08/329813,filed Oct. 11, 1994 and now U.S. Pat. No. 5,766,582.

BACKGROUND OF THE INVENTION

This invention relates to stable, aqueous solution formulations whichare free of products derived from human blood serum and which maintainhigh biological activity and high chemical and high physical stabilityof alfa-interferon for an extended period of time.

U.S. Pat. No. 4,496,537 discloses biologically stable alfa interferonaqueous solution formulations containing alfa interferon, human serumalbumin and alanine or glycine, water, and a buffer system to maintainthe pH at 6.5-8.0. The human serum albumin ("HSA") acts as a stabilizerfor alfa interferon and prevents losses of alfa interferon from solutionby coating and/or adsorption of the alfa interferon onto the stainlesssteel and glass surfaces of compounding vessels, process equipment andstorage containers. Solution formulations containing alfa interferon andHSA have maintained the chemical and biological stability of the alfainterferon when such solutions have been stored at 2-8° C. for extendedperiods, i.e., more than 2 years.

Recently, the worldwide AIDS epidemic has resulted in healthregistration agencies requiring manufacturers to place warnings onproducts, such as alfa interferon, which contain products derived fromhuman blood such as HSA.

There is a need to reformulate alfa-type interferon solution products toobtain a solution formulation free of human blood-derived products suchas HSA while maintaining high chemical, high physical stability and highbiological activity for alfa-type interferon in the aqueous solutionformulations for extended storage periods.

SUMMARY OF THE INVENTION

The present invention provides a stable, aqueous solution formulationwhich maintains high biological activity for alfa-type interferon and isfree of human blood-derived products which comprises:

a. 0.1×10⁶ to 100×10⁶ IU/mL of alfa-type interferon;

b. a buffer system to maintain a pH in the range of 4.5 to 7.1.

c. an effective amount of a chelating agent;

d. an amount of a sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivative sufficient to stabilize the alfa-typeinterferon against loss of alfa-type interferon;

e. an effective amount of a tonicity agent;

f. an effective amount of an antimicrobial preservative; and

g. an amount of water for injection sufficient to prepare a solution ofthe above-listed ingredients.

The present invention provides a stable, aqueous solution formulationhaving high alfa-type interferon biological activity and free of humanblood-derived products which comprises:

a. 0.1×10⁶ to 100×10⁶ IU/mL of alfa-type interferon.

b. a buffer system sufficient to maintain the pH of the solution in therange of 4.5 to 7.1;

c. about 0.01 to 1 mg/mL of disodium dihydrogenethylenediaminetetraacetate.

d. about 0.01 to 1 mg/mL of a sorbitan mono-9-octadecenoatepoly(oxy-1,2-ethanediyl) derivative;

e. about 1 to 9 mg/mL of sodium chloride;

f. an effective amount of an antimicrobial preservative selected fromm-cresol, phenol, methylparaben, propylparaben or mixtures thereof; and

g. water for injection q.s. ad. 1 mL.

In a preferred aspect, the present invention provides a stable, aqueoussolution formulation having high biological alfa-type interferonactivity and free of human blood-derived products, which comprises:

    ______________________________________                        mg/mL    ______________________________________    a.      Alfa-2 Interferon 5 × 10.sup.6 to                              50 × 10.sup.6 IU    b.      Sodium Phosphate Dibasic                              1.8            Anhydrous    c.      Sodium Phosphate Monobasic                              1.3            Monohydrate    d.      Disodium Dihydrogen Ethylene-                              0.1            diaminetetraacetate    e.      Polysorbate 80    0.1    f.      Methytparaben     1.2    g.      Propylparaben      0.12    h.      Sodium Chloride;  7.5            and    i.      Water for Injection                              q.s. ad 1 mL    ______________________________________

In another preferred aspect the present invention further provides astabile aqueous solution formulation having high biologicalalfa-interferon activity and free of human blood-derived products, whichcomprises:

    ______________________________________                       mg/mL    ______________________________________    a.      Alfa-Interferon  5 × 10.sup.6 to                             50 × 10.sup.6 IU    b.      Sodium Phosphate Dibasic                             1.8            Anhydrous    c.      Sodium Phosphate Monobasic                             1.3            Monohydrate    d.      Disodium Dihydrogen                             0.1            Ethylenediaminetetraacetate    e.      Polysorbate 80   0.1    f.      m-Cresol         1.5    g.      Sodium Chloride; 7.5            and    h.      Water for Injection                             q.s. ad 1 mL    ______________________________________

The present invention also provides a process of preparing a stable,aqueous solution formulation having high biological alfa-type interferonactivity and free of human blood-derived products comprising admixing aneffective amount of alfa-interferon with a buffer system capable ofmaintaining the pH within the range of 4.5 to 7.1, a chelating agent, asorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivative, atonicity agent, an antimicrobial preservative and water to form asolution. In a preferred aspect of the process of the present invention,the solution is prepared and maintained substantially free of dissolvedoxygen and a headspace of inert atmosphere above the solution ismaintained at a value of less than about 4% by volume of oxygen.

DETAILED DESCRIPTION

We have selected specific amounts of a specific set of ingredients thathave allowed us to develop an aqueous alfa-type interferon solutionformulation which does not contain human serum albumin yet maintainshigh chemical, biological and physical stability for the alfa-typeinterferon on storage at 2° to 8° C. for extended periods of at least 24months.

The term "free of human blood-derived products" as used herein inreference to the formulations of the present invention means that nohuman blood-derived products such as HSA are used in the preparation ofthe solution formulations of the present invention.

The term "high chemical stability" as used herein in reference to thealfa-interferon used in the formulations of the present invention meansthe alfa-interferon maintains at least 85%, preferably 85% to 100% ofits chemical integrity upon storage at 2° to 8° C. for at least 24months. See Tables 1 and 2. The chemical integrity is determined bymeasuring the protein content in an HPLC assay such as the one disclosedby T. L. Nagabhushan, et al., in an article entitled "Characterizationof Genetically Engineered ALFA-2 Interferon", pages 79-88 appearing inInterferon Research Clinical Application, and Regulatory Consideration,Zoon, et al., eds., Elsevier Science Publishing Co., Inc. 1984. (Seeresults in Tables 1 to 4).

The term "high biological stability" as used herein in reference to thealfa-interferon used in the formulations of the present invention meansthe alfa-interferon in the formulation maintains at least 75%,preferably at least 85%, more preferably 90% to 100% of its biologicalactivity upon storage at 2° to 8° C. for at least 24 months (see resultsin Tables 1 to 4) as measured in the standard method of inhibition ofthe cytopathic effect (CPE) of a virus such as the method disclosed byW. P. Protzman, et al., in J. Clinical Microbiology, (1985), 22,596-599.

The term "high physical stability" as used herein in reference to thealfa-interferon used in the formulations of the present invention meansthe formulation of the present invention remains clear, i.e., does notexhibit haze or visible particulate matter (i.e., particles greater thanabout 60 to 70 microns in diameter) on storage at 2° to 8° C. for atleast 24 months. See Tables 1, 2 and 3. The results listed in Tables 1,2 and 3 are surprising in that most solution formulations containingprotein products like alfa-interferon tend to develop visuallyobservable particulate matter (i.e., particles having diameters greaterthan 60 to 70 microns) upon extended storage even at 2° to 8° C. Thetest method used to determine particulate matter in the solutionformulation of this invention (see Tables 1 to 4) is described in TheUnited States Pharmacopeia/The National Formulary USP XXII/NF XVII,published by United States Pharmacopeial Convention, Inc., (1990),Rockville, Md.; see Physical Test <788> on pages 1596 to 1598. Themethod used to determine the visual description of the solutionformulations of this invention is also described in USP XXII as the"General Requirement Test and Assays <1> Injections" at pages 1470 to1472.

We have found that by adding a chelating agent to the formulations ofthe present invention, we have been able to avoid visible particulatematter. Typical suitable chelating agents include disodium dihydrogenethylenediamine tetraacetate (EDTA or edetate disodium) or citric acid.The use of edetate disodium is preferred. While we do not wish to bebound by any theory, it is believed that edetate disodium effectivelycomplexes with trace amounts of metal cations, such as Zn²⁺, Fe²⁺, Cu²⁺or Al³⁺, which ions may be present in excipients and packagingcomponents, e.g., rubber stoppers or gaskets. Since edetate disodium hasa higher affinity for these metal cations than the alfa-interferons, theinteraction between metal cations and alfa-interferon which results information of insoluble complexes (in the form of, for example, visibleparticulate matter) and loss of activity are avoided. The effectiveamount of the chelating agent is in the range of 0.01 to 1 mg/mL basedon 0.1×10⁶ to 100×10⁶ International Units ("IU") of alfa-typeinterferon/mL. Preferably, 0.1 mg of edetate disodium is used for 5×10⁶to 50×10⁶ IU of alfa-2 interferon.

The buffer systems suitable for the formulations of the presentinvention are those which maintain the pH of the aqueous solutionformulation in the range of 4.5 to 7.1, preferably 6.5-7.1 and mostpreferably 6.8. The use of a buffer system of sodium phosphate dibasicand sodium phosphate monobasic is preferred. Normally a 0.005 to 0.1molar buffer of the preferred sodium phosphate monobasic/dibasic buffersystem is used for a formulation containing 0.1×10⁶ to 100×10⁶ IU ofalfa-type interferon per mL. Other suitable buffer systems to maintainthe desired pH range of 4.5 to 7.1 include sodium citrate/citric acidand sodium acetate/acetic acid.

The tonicity agent useful in the present invention is any agent capableof rendering the formulations of the present invention iso-osmotic withhuman serum. Typical suitable tonicity agents include sodium chloride,mannitol, glycine, glucose and sorbitol. Use of sodium chloride as atonicity agent is preferred.

The amount of the tonicity agent used is in the range of 1 to 10 mg/mLwhen the formulation of the present invention contains 0.1×10⁶ to100×10⁶ IU of alfa-interferon/mL. The use of 7.5 mg/mL of sodiumchloride is preferred for 5×10⁶ to 50×10⁶ IU of alfa-type interferon permL in the formulations of the present invention.

The sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivativessuch as polysorbate 80 or polysorbate 20 are useful as a stabilizingagent to prevent adsorption of the alfa-interferon proteins such asalfa-2b interferon onto the stainless steel and glass surfaces of theequipment used to make the indictable formulations containing alfa-typeinterferon. The amount of polysorbate 20 or 80 effective in theformulation of this invention is in the range of 0.01 to 1.0 mg per mLfor a formulation containing 0.1×10⁶ to 100×10⁶ IU of alfa-interferonper mL. The use of polysorbate 80 is preferred. The use of 0.1 mg/mL ofpolysorbate 80 is more preferred in all the solution formulations of thepresent invention. When the concentrations of alfa-interferon such asalfa-2 interferon is less than about 15×10⁶ IU/mL, e.g., 6×10⁶ IU/mL,loss of activity due to adsorption of the alfa interferon in the absenceof polysorbate 80 significantly lowers the biological activity of theformulation. Surprisingly, we have found that polysorbate 80 preventsloss of alfa-2b interferon and allows systemic delivery of the alfa-2binterferon without loss of biological activity. In the course ofdevelopment of the formulation of the present invention, we surprisinglyfound that polysorbate 80 provided superior chemical and biologicalstability to alfa-2b interferon compared to other non-ionic surfactants,e.g., Pluronic F127 and Pluronic F-68.

The amount of alfa-interferon useful in the formulation of the presentinvention is in the range of 0.1×10⁶ to 100×10⁶ IU/mL, preferably 5×10⁶to 50×10⁶ IU/mL.

The term "alfa-interferon" as used herein means the family of highlyhomologous species-specific proteins that inhibit viral replication andcellular proliferation and modulate immune response. Typical suitablealfa-interferons include interferon alfa-2a such as ROFERON A interferonalfa-2a available from Hoffmann-La Roche, Nutley, N.J., interferonalfa-2b such as INTRON A interferon alfa-2b available from ScheringCorporation, Kenilworth, N.J. interferon alfa-2c such as BEROFORinterferon alfa-2c available from Boehringer Ingelheim Pharmaceutical,Inc., Ridgefield, Conn., interferon alfa-n1, a purified blend of naturalalfa interferons such as SUMIFERON available from Sumitomo, Japan or asWELLFERON interferon alfa-n1 available from The Wellcome FoundationLtd., London, Great Britain, or consensus alfa interferon available fromAmgen, Inc., Newbury Park, Calif., or interferon alfa-n3, a mixture ofnatural alfa interferons, made by Interferon Sciences and available fromthe Purdue Frederick Co., Norwalk, Conn., under the ALFERON tradename.The use of interferon alfa-2a or alfa-2b is preferred. The use ofinterferon alfa-2b is more preferred.

The antimicrobial preservatives found useful in the present inventioninclude m-cresol, phenol and methyl-paraben and propylparaben andmixtures of the above-listed preservatives, e.g., phenol-methylparabenmixtures. The effective amount of m-cresol found useful in the presentinvention is in the range of 0.5 to 2 mg/mL for a formulation containing0.1×10⁶ to 100×10⁶ IU/mL of alfa-interferon. It is preferred to use 1.5mg/mL of m-cresol for a formulation containing 5×10⁶ to 50×10⁶ IU/mL ofinterferon alfa-2b.

The effective amount of phenol found useful is in the range of 0.5 to 5mg/mL for a solution formulation containing 0.1×10⁶ to 100×10⁶ IU/mL ofalfa-interferon.

The effective amount of methylparaben is in the range of 0.6 to 1.8mg/mL and the amount of propylparaben is in the range of 0.06 to 0.18mg/mL when the formulation of the present invention contains 0.1×10⁶ to100×10⁶ IU/mL of alfa-interferon.

It is preferred to use 1.2 mg/mL of methylparaben in combination with0.12 mg/mL of propylparaben when the formulation of the presentinvention contains 0.1×10⁶ to 100×10⁶ IU/mL of alfa-2b interferon.

The use of m-cresol as an antimicrobial preservative is more preferred.

The water used for preparation of the formulations of the presentinvention is preferably water for injection.

During the course of development of the aqueous solution formulations ofthe present invention that would maintain high biological activity aswell as high chemical and high physical stability of the alfa-interferonover an extended storage period without employing HSA as a stabilizer,we identified that the amount of a sorbitan mono-9-octadecenoatepoly(oxy-1,2-ethanediyl) derivative such as polysorbate 80 required toact as a stabilizing agent for the alfa-interferon had a direct effecton the effective amount of the antimicrobial preservative which could beadded to the aqueous solution formulation to provide the appropriateantimicrobial protection for said formulation pursuant to variousworldwide health registration requirements without causing undesirablehaze formation in the solution.

Thus, when the preferred stabilizing agent, polysorbate 80, was presentin formulations of the present invention, in the preferred effectiveamount of 0.1 mg/mL, the effective amount of the preferred antimicrobialpreservative, e.g., m-cresol which could be added without causing hazingof said formulation was found to be critical. For example if the amountof m-cresol added to a formulation which contained 0.1 mg/mL ofpolysorbate 80, such as shown in Example 3, is increased to greater than1.75 mg/mL, hazing was observed. A similar hazing problem was observedwhen the amount of polysorbate 80 in the resultant formulation wasvaried from 0.01 to 1 mg/mL. No hazing was observed when 1.75 mg/mL orless, preferably about 1.5 mg/mL of m-cresol was added to a formulationprepared in accordance with the procedures of Example 3 which contains0.1 mg/mL of polysorbate 80. This criticality was also observed with theparabens and phenol when they were used as antimicrobial preservatives.For formulations of the present invention containing 0.01 to 1 mg/mL ofpolysorbate 80, the effective amount of methylparaben should be no morethan about 1.2 mg/mL when used with 0.12 mg/mL of propylparaben to avoidhazing, and the effective amount of phenol (when it is used in place ofthe parabens) should be in the range of 0.5 to less than about 4 mg/mLto avoid hazing.

Alfa-interferon formulations are useful for treatment of a variety ofdisease states such as renal cell carcinomas, AIDS-related Kaposi'ssarcoma, chronic and acute hepatitis B, chronic and acute non-A, non-B/Chepatitis. The formulations of the present invention are useful intreating these disease states preferably as injectable aqueoussolutions.

EXAMPLES

The following non-limiting examples illustrate the preparation of theaqueous solutions of alfa-interferons.

The procedures listed after Example 5 are used to prepare theformulations of the present invention of Examples 1 to 5.

Example 1

    ______________________________________    ActiveSubstance:                  Interferon alfa-2b                                0.1 × 10.sup.6 - 100 x                                10.sup.6 IU/mL*    Buffer:       Sodium Phosphate                                0.005-0.1 M                  (monobasic/dibasic)    Chetating Agent:                  Edetate Disodium                                0.01-1 mg/mL    Stabilizer:   Polysorbate 80                                0.01-1 mg/mL    Tonicity Adjusting    Agent:        Sodium Chloride                                1-9 mg/mL    Antimicrobial    Preservative: m-Cresol      0.5-1.75 mg/mL                  or Phenol     0.5-<4 mg/mL                  or Methylparaben                                0.6-1.2 mg/mL                  Propylparaben 0.06-0.12 mg/mL    Solvent:      Water for Injection                                q.s. ad 1 mL    ______________________________________     *IU--International Units

Example 2

    ______________________________________    interferon alfa-2b      10 × 10.sup.6 IU/mL    Sodium Phosphate Dibasic Anhydrous                            1.8 mg/mL    Sodium Phosphate Monobasic Monohydrate                            1.3 mg/mL    Edetate Disodium        0.1 mg/mL    Polysorbate 50          0.1 mg/mL    Methylparaben           1.2  mg/mL    Propylparaben           0.12 mg/mL    Sodium Chloride         7.5 mg/mL    Water for Injection q.s. ad                            1mL    ______________________________________

Example 3

    ______________________________________    interferon alfa-2b      10 × 10.sup.6 IU/mL    Sodium Phosphate Dibasic Anhydrous                            1.8 mg/mL    Sodium Phosphate Monobasic Monohydrate                            1.3 mg/mL    Edetate Disodium        0.1 mg/mL    Polysorbate 50          0.1 mg/mL    m-Cresol                1.5 mg/mL    Sodium Chloride         7.5 mg/mL    Water for Injection q.s. ad                            1 mL    ______________________________________

Example 4

The formulation of Example 3 was prepared with 6×10⁶ IU/mL of alfa-2binterferon in accordance with the method of manufacture detailed hereinbelow using nitrogen sparging of the solution and maintaining no morethan about 4% by volume of oxygen in the headspace.

Vials containing a label volume of 3 mL of solution were stored at 30°,25° and 4° C. The results are summarized in Table 3.

Example 5

The formulation of Example 4 was prepared in accordance with the methodof manufacture detailed hereinbelow in all details except no nitrogenwas sparged through the solution or overlaid upon it and the oxygenvolume in the headspace was ˜20% by volume as found in ambient air.

Vials containing a label volume of 3 mL of solution were stored at 30°,25° and 4° C. The results are summarized in Table 4.

Similar results are expected if the interferon alfa-2b in Examples 1 to5 is substituted by an equivalent amount of Roferon A, Wellferon orSumiferon interferon alfa.

                                      TABLE 1    __________________________________________________________________________    Interferon Alfa-2b Stability Data on Example 2           Anti-Viral Assay                       Protein Content                                  Particulate Matter    Time        Temp           (CPE)       (HPLC Assay)                                  (particles/container)    (month)        (° C.)           (× 10.sup.6 IU/mL)                  (% L.S.)                       (mcg/mL)                            (% of Initial)                                  ≧10μ                                     ≧25μ                                        ≧50μ                                           Description    __________________________________________________________________________    Initial 10.0  100  42.5 100   40 3  1  ccs*     3  4  9.0     90  42.4 100   16 13 11 ccs     6  4  10.0   100  41.8  98   8  3  1  ccs     9  4  10.0   100  43.3 102   52 3  0  ccs    12  4  10.0   100  44.3 104   17 4  2  ccs    18  4  9.8     98  41.5  98   6  1  0  ccs    24  4  10.0   100  39.5  93   5  1  0  ccs    __________________________________________________________________________     *ccs  clear, colorless solution, essentially free of visible particles.

                                      TABLE 2    __________________________________________________________________________    Interferon Alfa-2b Stability Data on Example 3                       Protein Content                                  Particulate Matter    Time        Temp           Anti-Viral Assay                       (HPLC Assay)                                  (particles/container)    (month)        (° C.)           (× 10.sup.6 IU/mL)                  (% L.S.)                       (mcg/mL)                            (% of Initial)                                  ≧10μ                                     ≧25μ                                        ≧50μ                                           Description    __________________________________________________________________________    Initial 10.3  103  40.3 100   68 4   3 ccs*    1   4   10    100  40.6 101   142                                     24 23 ccs    3   4   10    100  41.4 103   311                                     63 35 ccs    6   4   10    100  42.5 105   206                                     17 16 ccs    __________________________________________________________________________     *ccs  clear, colorless solution, essentially free of visible particles.

                                      TABLE 3    __________________________________________________________________________    Interferon Alfa 2b Stability Data on Example 4               Anti-Viral Assay                           Protein Content       Particulate Matter    Time        Temp           Position               (CPE)       (HPLC Assay)                                      m-Cresol Assay                                                 No. of particles/vial    (month)        (° C.)           of Vial*               (× 10.sup.6 IU/mL)                      (% L.S.)                           (mcg/mL)                                (% of Initial)                                      (mg/mL)                                           % LS                                              pH ≧10                                                     ≧25                                                         ≧50                                                             Description    __________________________________________________________________________    Initial    6.48   108  25.7 100   1.47 98.0                                              6.91                                                 23  2   0   CCS*    1   30 UP  6.00   100  24.8 96.5  1.47 98.0                                              6.90                                                 109 61  16  CCS           INV 6.00   100  24.8 96.5  1.47 98.0                                              6.90                                                 55  11  2   CCS    3    4 UP  6.00   100  23.5 91.4  1.46 97.3                                              6.88                                                 29  2   0   CCS           INV 6.00   100  24.2 94.2  1.48 98.7                                              6.87                                                 59  23  2   CCS    3   25 UP  6.00   100  21.7 84.4  1.43 95.3                                              6.88                                                 141 76  17  CCS           INV 6.00   100  21.7 84.4  1.47 98.0                                              6.88                                                 49  16  3   CCS    __________________________________________________________________________     *UP  Upright     INV  Inverted     *CCS  Clear, colorless solution, essentially free of visible particles.

                                      TABLE 4    __________________________________________________________________________    Interferon Alfa 2b Stability Data on Example 5               Anti-Viral Assay                           Protein Content       Particulate Matter    Time        Temp           Position               (CPE)       (HPLC Assay)                                      m-Cresol Assay                                                 No. of particles/vial    (month)        (° C.)           of Vial               (× 10.sup.6 IU/mL)                      (% L.S.)                           (mcg/mL)                                (% of Initial)                                      (mg/mL)                                           % LS                                              pH ≧10                                                     ≧25                                                         ≧50                                                             Description    __________________________________________________________________________    Initial    6.00   100  25.5 100   1.47 98.0                                              6.85                                                 144 4   4   CCS*    1   30 UP  6.00   100  19.5 76.5  1.49 99.3                                              6.82                                                 89  3   1   CCS           INV 6.00   100  19.5 76.5  1.50 100                                              6.83                                                 64  1   0   CCS    3    4 UP  6.00   100  24.0 94.1  1.43 95.3                                              6.81                                                 39  1   0   CCS           INV 6.00   100  24.0 94.1  1.43 95.3                                              6.82                                                 77  19  6   CCS    3   25 UP  6.00   100  20.2 79.2  1.39 92.7                                              6.82                                                 57  1   0   CCS           INV 6.00   100  19.6 76.9  1.4i 94.0                                              6.82                                                 140 24  1   CCS    __________________________________________________________________________     *CCS Clear, colorless solution, essentlally free of visible particles.

Method of Manufacture for Examples 1 to 5

A. Compounding Paraben-Containing Aqueous Solution Formulations Such asShown in Example 2

1. Charge approximately 80% of the water for injection at a temperaturegreater than 70° C. into a suitable jacketed compounding vessel equippedwith an agitator.

2. Separately charge approximately 30% of the water for injection intoanother suitable vessel. Cool and maintain the water temperature between20° and 25° C. Begin sparging and overlaying the water which will beused to bring the batch to final volume with filtered nitrogen tomaintain a dissolved oxygen level at or below 0.25 ppm.

3. Charge and dissolve with mixing methylparaben and propylparaben intothe compounding vessel in step 1 while maintaining the temperature ofthe solution between 70° and 80° C.

4. Cool the solution in step 3 to a temperature between 20° and 25° C.Sparge and overlay the solution with filtered nitrogen. Maintain adissolved oxygen level at or below 0.25 ppm.

5. Charge and dissolve with mixing the following ingredients into thesolution in step 4 while maintaining nitrogen sparging and overlaying:

Sodium phosphate dibasic anhydrous

Sodium phosphate monobasic monohydrate

Edetate disodium

Sodium chloride

6. Discontinue nitrogen sparging of the solution from step 5. Maintainnitrogen overlaying in the compounding vessel.

7. Charge and dissolve polysorbate 80 in approximately 50 mL of waterfor injection (for a 1-liter size batch) in a separate vessel. Transferthe polysorbate 80 solution into the solution from step 6.

8. Check the pH of the solution. It should be between 6.6 and 7.0. No pHadjustment is required.

9. Charge interferon alfa-2b bulk drug solution into the solution instep 8 while mixing.

10. Add water for injection that has been sparged with nitrogen (fromstep 2) to bring batch to final volume. Agitate solution gently untilhomogeneous.

11. Aseptically filter the solution through a sterilized filter that hasbeen washed and tested for integrity. Collect the sterilized solutioninto a sterilized filling vessel that has been overlaid withsterile-filtered nitrogen. Integrity test the filter after filtration.

12. Overlay filling vessel in step 11 with sterile-filtered nitrogen andseal.

B. Compounding m-Cresol-Containing Aqueous Solution Formulations Such asShown in Example 3

The manufacturing procedure used to prepare the aqueous solutionscontaining m-cresol as a preservative (such as shown in Example 3) isexactly the same as described hereinabove except the temperature of thesolution in Step 3 is maintained between 20° and 25° C. and the m-cresolis charged after Step 6.

C. Compounding HSA-Free Aqueous Alfa Interferon Solution FormulationsUnder Ambient Air

The manufacturing procedure used to prepare the HSA-free aqueous alfainterferon formulations of Examples 1 to 4 was used to prepare theformulations such as that of Example 5 except that all the steps wereperformed under ambient air; no nitrogen was sparged through thesolution or overlaid upon it and ambient air (normally containing about20% by volume of oxygen) occupied the headspace volume.

To maintain high chemical, physical and biological stability, it ispreferred that the water used to prepare the aqueous alfa interferonsolution as well as the so-formed aqueous alfa interferon solution besubstantially free of dissolved oxygen, and the aqueous solution be madeand stored with a headspace of an inert atmosphere, such as nitrogen,containing no more than about 4% volume of oxygen. By the term"substantially free of dissolved oxygen" as used herein is meant anoxygen level of no more than about 0.25 ppm at a water temperature ofabout 20°-25° C. Normally, this preferred dissolved oxygen level of 0.25ppm is conveniently achieved by sparging an inert atmosphere, e.g.nitrogen gas into the water used to prepare the aqueous solutions(maintained at a temperature of about 20°-25° C.) for a time sufficient(e.g. about 30 minutes) to lower the dissolved oxygen to a value of nomore than about 0.25 ppm. The sparging is continued throughout themanufacturing procedure to maintain the dissolved oxygen level at 0.25ppm. We have found that aqueous formulations of the present inventionwhich have a dissolved oxygen level of 1 ppm and an oxygen content inthe headspace of 7% by volume demonstrated significantly greater loss ofchemical stability of the alfa interferon after 3 months of storage at25° C. compared to a similar aqueous formulation having the preferreddissolved oxygen level of 0.25 ppm and an oxygen content in theheadspace of 4% by volume stored under the same conditions.

A side-by-side comparison of the alfa-2b interferon solution stabilitydata shown in Tables 3 and 4 shows that there is no significantstability difference between the aqueous solution formulations of thepresent invention which were prepared under the nitrogen/low oxygenconditions used in Example 4 and those prepared in accordance withExample 5 under ambient air during storage for 3 months at 4° C. Incontrast, a comparison of the alfa-2b interferon stability in solutionsof Examples 4 and 5 stored at higher temperatures, e.g, 25° and 30° C.shows the protective effect achieved by the preferred (safer) use ofnitrogen sparging to effect low dissolved oxygen levels in the aqueoussolution while simultaneously maintaining an oxygen content in theheadspace at a value of no more than about 4% by volume.

The aqueous solution formulation of the present invention may be storedin any suitable washed and sterilized filing vessels or container suchas 2-mL or 5-mL Type I flint glass vials stoppered with gray butylrubber closures. The aqueous solution formulations of the presentinvention may also be stored in prefilled multi-dose syringes such asthose useful for delivery of indictable solutions of drugs such asinsulin. Typical suitable syringes include systems comprising aprefilled vial attached to a pen-type syringe such as the Novolet NovoPen available from Novo Nordisk. Typical suitable systems include aprefilled, pen-type syringe which allows easy self-injection by the useras well as accurate, reproducible dose settings.

The aqueous solutions formulations of the present invention, such aspresent in the Examples may also be lyophilized to form a powder forreconstitution. The lyophilized alfa-type interferon powder is expectedto maintain its chemical and biological stability when stored at 2° to8° C. for at least 2 years.

We claim:
 1. A stable, aqueous formulation having at least 75% of theinitial biological activity for alfa interferon and free of humanblood-derived products which consists essentially of:a. 0.1×10⁶ to100×10⁶ IU/mL of alfa-interferon; b. a buffer system to maintain a pH inthe range of 4.5 to 7.1. c. an effective amount of edetate disodiumdihydrogen ethylenediamine tetraacetate as a chelating agent sufficientto avoid visible particulate matter; d. an amount of a sorbitanmono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivative sufficient tostabilize the alfa-type interferon against loss of alfa-type interferonbiological activity; e. an effective amount of a tonicity agentsufficient to render said formulation iso-osmotic with human serum; f.an effective amount of an antimicrobial preservative selected fromm-cresol, phenol, methylparaben, propylparaben or mixtures thereofsufficient to provide appropriate antimicrobial protection for saidformulation without causing undesirable hazing formation; and g. anamount of water for injection sufficient to prepare a solution of theabove-listed ingredients.
 2. The composition of claim 1 wherein thebuffer system is sodium dibasic phosphate and sodium monobasicphosphate.
 3. The composition of claim 1 wherein the chelating agent isedetate disodium or citric acid.
 4. The composition of claim 1 whereinthe tonicity agent is sodium chloride.
 5. The composition of claim 1wherein the alfa-interferon is interferon alfa-2.
 6. The stable, aqueousformulation of claim 1 wherein the buffer system is sodiumcitrate/citric acid.
 7. A stable, aqueous formulation having at least75% of the initial alfa interferon biological activity and substantiallyfree of human blood-derived products which consists essentially of:a.0.1×10⁶ to 100×10⁶ IU/mL of alfa interferon. b. a buffer systemsufficient to maintain the pH of the solution in the range of 4.5 to7.1; c. about 0.01 to 1 mg/mL of edetate disodium dihydrogenethylenediaminetetraacetate; d. about 0.01 to 1 mg/mL of a sorbitanmono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivative; e. about 1 to9 mg/mL of sodium chloride; f. an effective amount of an antimicrobialpreservative selected from m-cresol, phenol, methylparaben,propylparaben or mixtures thereof sufficient to provide appropriateantimicrobial protection for said formulation without causingundesirable hazing formation; and g. a quantity of water for injectionsufficient to make a 1 mL solution of the above-listed ingredients. 8.The composition of claim 7 wherein interferon alfa-2b is used.
 9. Thecomposition of claim 7 wherein interferon alfa-2a is used.
 10. Thecomposition of claim 7 wherein said preservative is m-cresol.
 11. Thecomposition of claim 7 wherein said preservative is a mixture ofmethylparaben and propylparaben.
 12. The stable aqueous formulation ofclaim 7 wherein the buffer system is sodium citrate/citric acid.
 13. Astable aqueous solution formulation having at least 75% of initialbiological activity of interferon alfa-2 and free of human serumalbumin, which consists essentially of:

    ______________________________________                         mg/mL    ______________________________________    a.      Interferon alfa-2  5 × 10.sup.6 to                               50 × 10.sup.6 IU/mL    b.      Sodium Phosphate Dibasic                               1.8            Anhydrous    c.      Sodium Phosphate Monobasic                               1.3            Monohydrate    d.      Disodium Dihydrogen                               0.1            Ethylenediamine tetraacetate    e.       A! The Sorbitan Mono-9-                               0.1            Octadecenoate Poly(Oxy-1,2-            Ethanediyl) Derivative,            polyoxyethylene (20) sorbitan            mono-oleate    f.      Methylparaben      1.2    g.      Propylparaben       0.12    h.      Sodium Chloride; and                               7.5    i.      A sufficient quantity of Water for            Injection to make a  q.s. ad! 1 mL            solution of the above-listed            ingredients    ______________________________________


14. The formulation of claim 13 wherein interferon alfa-2b is used. 15.A stabile, aqueous solution formulation having at least 75% of theinitial biological activity for interferon alfa-2 and free of humanserum albumin, which consisting essentially of:

    ______________________________________                         mg/mL    ______________________________________    A.     Interferon alfa-2   5 × 10.sup.6 to                               50 × 10.sup.6 IU/mL    B.     Sodium Phosphate Dibasic                               1.8           Anhydrous    C.     Sodium Phosphate Monobasic                               1.3           Monohydrate    D.     Disodium Dihydrogen 0.1           Ethylenediaminetetraacetate    E.      A! The Sorbitan Mono-9-                               0.1           Octadecenoate PoIy(Oxy-1,2-           Ethanediyl) Derivative,           polyoxethylene (20) sorbitan mono-           oleate    F.     m-Cresol            1.5    g.     Sodium Chloride; and                               7.5    h.     a sufficient quantity of Water for           Injection to make a  q.s. ad! 1 mL           solution of the above-listed           ingredients    ______________________________________


16. The formulation of claim 15 wherein interferon alfa-2a is used. 17.The formulation of claim 15 wherein interferon alfa-2b is used.
 18. Astable, aqueous formulation having at least 75% of initial alfainterferon biological activity and free of human blood-derived productsand manitol as a bulking agent which comprises about 0.1×10⁶ to 100×10⁶/U/mL of alfa interfferon and about 0.01. to 1 mg/mL of a chelatingagent effective to avoid formation of visible particulate matter in theformulation.
 19. The stable aqueous formulation of claim 18 wherein thechelating agent is edetate disodium.
 20. A method of avoiding visibleparticulate matter formation in an aqueous alfa interferon formulationhaving at least 75% of the initial biological activity of the alfainterferon and free of human blood-derived products which comprisesadmixing an aqueous alfa interferon formulation with an amount of achelating agent effective to avoid formation of visible particulatematter in the formulation.
 21. The method of claim 20 wherein the amountof alfa interferon is about 0.1×10⁶ to 100×10⁶ IU/mL.
 22. The method ofclaim 20 wherein the chelating agent is edetate disodium.
 23. The methodof claim 20 wherein the alfa interferon is inteferon alfa-2a.
 24. Themethod of claim 20 wherein the alfa interferon is interferon alfa-2b.